There has been significant recent progress in our understanding of the molecular mechanisms of thyroid hormone (TH) action, with the description of the thyroid hormone response element (TRE) and the thyroid hormone receptors (TRs). For instance, it is known that TRs can function as heterodimers with other nuclear proteins such as retinoid X receptors (RXRs). A major question, however, remains. How does TH, and its interactions with bound TRs and RXRs, cause the activation of the basal transcriptional machinery in the regulation of gene expression? Is the communication direct and/or indirect via TR/RXR adaptor molecules (TRAMS)? There is already evidence for the former; however, coactivators, mediators or repressors may play roles in the latter. The overall goal of this application is to determine the molecular mechanisms involved in TH- regulated transactivation of responsive genes. We hypothesize that the TH-mediated, TR-dependent transactivation of gene expression involves indirect as well as direct interactions with the basal transcriptional machinery. The specific aims of this proposal are: (1) to utilize a TH- responsive cell-free in vitro transcription system to provide additional evidence that TR/RXR adaptor molecules (TRAMS), other than TRs and RXRs, are involved in TH-stimulated gene expression; (2) to identify TRAMS as proteins that interact with either TR, RXR or TR/RXR heterodimer using coimmunoprecipitation techniques, including the use of isoform-specific TR and RXR antibodies, affinity chromatography and far western blotting; (3) to obtain cDNAs encoding TRAMs, possibly coactivators or repressors, using either a biochemical approach, and/or far western and in vivo genetic screens; and (4) to characterize and test the ability of these TRAMS to function as coactivators or repressors, using in vitro transcription and transfection systems. It is expected that a full understanding of the molecular players and events in the TH-mediated activation of genes will advance our knowledge of the role of TH in health and disease, including inherited resistance to TH and variable tissue responses to TH in humans.